BIOLOGY OF SODIUM BICARBONATE TRANSPORT

描述(摘自申请者摘要)：碳酸氢钠 肾脏中的辅助转运蛋白有助于维持全身 酸碱平衡。这些完整的膜蛋白发挥着重要的作用 在调节细胞内pH和调节跨上皮钠和 碳酸氢盐的运输。我们对其功能特点的理解 在最近克隆了几个成员后，这些转运蛋白得到了改善 属于碳酸氢钠共转运(NBC)家族。我们最近克隆了 具有功能特征的NBC3，是NBC家族中独特的成员 二苯乙烯不敏感，电子中和，EIPA可抑制。在肾脏，NBC3是 在A型间质细胞中分选到顶膜和 B型间质细胞的基底膜。此外，NBC3 与空泡H+-ATPase共定位和免疫共沉淀 共识PDZ-相互作用域，分别为-ETAL和-DTAL，这可以 在膜分离中起着重要的作用。在此基础上，我们筛选出一名人类 以NBC3 C末端为基础的酵母双杂交构建肾脏cDNA文库 诱饵，并分离出一种新的蛋白NBC3BP，它具有三个潜在的功能 PDZ结合域。NBC3BP定位于A型根尖膜 并与来自肾脏的NBC3免疫共沉淀。我们 假设液泡H+-ATPase的NBC3和58kD亚基被分类为 A型间质细胞的顶膜，通过它们的相互作用 C-末端PDZ-与PDZ结合蛋白NBC3BP相互作用的结构域 在该细胞类型的顶膜上表达，而在B型细胞中不表达 嵌插细胞。我们将使用MDCK I细胞系，它对NBC3BP和 以NBC3为模型的A型类似于根尖膜的间质细胞 实现本提案目标的系统。成功完成这项工作 该项目将加强我们对相关分子机制的理解 用于确定酸碱转运体的极性。

DESCRIPTION (Adapted from the Applicant's Abstract): Sodium bicarbonate cotransporters in the kidney contribute to the maintenance of whole body acid-base homeostasis. These integral membrane proteins play an important role in regulating intracellular pH and in mediating transepithelial sodium and bicarbonate tranport. Our understanding of the functional characteristics of these transporters has improved following the recent cloning of several members of the sodium bicarbonate cotranport (NBC) family. We have recently cloned and functionally characterized NBC3, a unique member of the NBC family which is stilbene-insensitive, electroneutral, and EIPA-inhibitable. In kidney, NBC3 is sorted to the apical membrane in type A intercalated cells and to the basolateral membrane in type B intercalated cells. Furthermore, NBC3 co-localized and co-immunoprecipitated with the vacuolar H+-ATPase have consensus PDZ-interacting domain, -ETAL and -DTAL respectively, which could play an important role in membrane sorting. On this basis, we screened a human kidney cDNA library in a yeast two-hybrid assay using the C-terminus of NBC3 as bait, and isolated a novel protein, NBC3BP, which has three potential PDZ-binding domains. NBC3BP was localized to the apical membrane of type A intercalated cells and was co-immunoprecipitated with NBC3 from kidney. We hypothesize that NBC3 and 58 kD subunit of the vacuolar H+-ATPase are sorted to the apical membrane of type A intercalated cells, via interaction of their C-terminal PDZ-interacting domains with the PDZ-binding protein, NBC3BP, which is expressed on the apical membrane of this cell type and not in type B intercalated cells. We will use the MDCK I cell line which sorts NBC3BP and NBC3 to the apical membrane similar to the type A intercalated cell as a model system for achieving the goals of this proposal. Successful completion of this project will enhance our understanding of the molecular mechanisms responsible for determining the polarity of acid-base transporters.